- Short report
- Open Access
Extended blood circulation and joint accumulation of a p(HPMA-co-AzMA)-based nanoconjugate in a murine model of rheumatoid arthritis
Molecular and Cellular Therapies volume 2, Article number: 29 (2014)
We recently synthesized a hydrophilic polymer, poly(N-(2-hydroxypropyl)methacrylamide-co-N-(3-azidopropyl)methacrylamide), p(HPMA-co-AzMA), by RAFT polymerization using a novel azide-containing methacrylamide monomer that through a post modification strategy using click chemistry enabled facile preparation of a panel of versatile and well-defined bioconjugates. In this work we screen a panel of different molecular weight (Mw) fluorescently tagged p(HPMA-co-AzMA) in healthy mice, by live bioimaging, to select an extended circulatory half-life material for investigating joint accumulation in a murine collagen antibody-induced arthritis model.
Fluorescence image analysis revealed half-lifes of <20 min, 2.8 h and 6.4 h for p(HPMA-co-AzMA) of 15, 36 and 54 kDa, respectively, with ~10% polymer retained in the blood after 24 h for the highest Mw. p(HPMA-co-AzMA) of 54 kDa showed enhanced accumulation in the joints of the arthritic mouse model with a bioavailability (AUC = 1783% · h) ~12 times higher (P = 0.01) than healthy control (AUC = 148% · h).
p(HPMA-co-AzMA) of 54 kDa exhibited extended circulatory half-life and preferential accumulation in inflamed joints of a murine model of rheumatoid arthritis (RA). This combined with well-defined polymer size and versatility for conjugation of a range of biomolecules promotes p(HPMA-co-AzMA) for potential applications in the delivery of drugs for treatment of RA.
Rheumatoid arthritis (RA) is a chronic progressive autoimmune disease affecting ~ 1% of the population causing cartilage and bone destruction in synovial joints . The pathogenesis involves synovial infiltration by circulatory immune cells that induce inflammation, modulated predominantly by proinflammatory cytokines such as tumor necrosis factor alfa (TNFα) [2, 3]. Common clinical treatments include nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and disease-modifying antirheumatic drugs (DMARDs) such as anti-TNFα antibodies. Although effective in many patients, these therapeutics have associated side-effects such as myelosuppression and increased infection risk, primarily due to a general systemic immune suppression that necessitates alternative approaches [1, 4]. RA associated angiogenesis, necessary for formation of pannus and invasion of inflammatory cells into the synovial tissue, has been identified to establish a macromolecular retention effect [5–7], conceptually similar to the EPR effect for tumors . Hydrophilic polymers synthesized with high molecular weights (Mw) enable the construction of polymer macromolecular drugs  with extended blood circulation and specific targeting to arthritic joints. This has been used for local suppression of proinflammatory cytokines such as TNFα  that may increase the clinical efficacy and reduce generalized side effects.
The field of polymer therapeutics requires increased attention to defining and minimizing macromolecule heterogeneity and polydispersity due to its great influence on pharmacokinetics, safety and efficacy of polymer therapeutics and to identify precise polymer characteristics required for delivery e.g. to RA affected tissue [11, 12]. We have recently synthesized a novel azide containing copolymer, poly(N-(2-hydroxypropyl)methacrylamide-co-N-(3-azidopropyl)methacrylamide), (p(HPMA-co-AzMA)), through a versatile post-modification procedure to produce a panel of well-defined polymer bioconjugates of narrow PdI (Figure 1) . Compared to existing bioconjugation strategies for RA [14, 15], this may provide superior polymer characteristics in terms of flexibility for modification and low polydispersity.
We hypothesize that the p(HPMA-co-AzMA) constructs will exhibit prolonged blood circulation and specific accumulation in inflamed joints in a murine model of rheumatoid arthritis supporting its potential therapeutic application for RA.
For blood circulation studies, a PBS solution (100 μL) of p(HPMA-co-AzMA) polymers (4 mg/kg) of different Mw (15, 36 and 54 kDa) labeled with an Atto680 dye (ATTO-TEC GmbH, Siegen, Germany), or PBS only control was administered intravenously (i.v.) in healthy BALB/c male mice (8 week-old, Taconic Europe, Ry, Denmark). Blood volumes (~80 μL, max 2 pr. animal) were sampled from 5 min up to 24 h post-injection and the blood plasma was subsequently transferred to narrow 20 uL glass capillary tubes (minicaps, Hirschmann) and imaged with the IVIS® Spectrum using a 675/720 nm filterset and analyzed using the Living Image software version 4.3 (PerkinElmer).
Nine week-old, male, DBA/1 (Taconic Europe, Ry, Denmark) were used for the collagen antibody-induced arthritis (CAIA) model  with intraperitoneal (i.p.) injection of the monoclonal antibody mix (Arthrogen-CIA® arthritogenic monoclonal antibody 5 clones cocktail kit, Chondrex, Inc. Redmond, USA) and a subsequent LPS injection 3 days later. The severity of the arthritic condition was monitored by daily clinical scoring of joints (1–4)  with symptoms developing ~ day 3 and peaking around day 8. Healthy control mice (DBA/1) were injected i.p. with PBS.
For the joint accumulation studies, a PBS solution of the 54 kDa Atto680-labeled (0.57 w/w%) p(HPMA-co-AzMA), or the free dye, was administered by intravenous (i.v.) injection to arthritic and control animals at a polymer dose of 4 mg/kg or the dye-equivalent amount (22.6 ug/kg) of free dye. This dose was comparable to existing studies [17, 18] and at a concentration by which the polymer conjugated dye could be easily visualized using in vivo fluorescence imaging. Joint fluorescence was monitored before, and up to, 24 h post-injection by IVIS Spectrum in vivo imaging of isoflurane (2.5%) anesthetized mice. Multispectral fluorescence images covering the absorption-emission profile of the Atto680 dye were acquired using the Living Image software (PerkinElmer). Images were spectrally unmixed to subtract background fluorescence, which enabled the total fluorescence emission from joint areas from each mouse to be quantified. Measured intensities of the fluorescence emission are generally linear dependent on fluorophore concentration, extinction coefficient and quantum yield, which enables a direct correlation to polymer levels in the blood or tissue .
All image data was analyzed using Prism (GraphPad Software Inc.). Blood polymer levels were normalized to the value measured at 5 min and joint polymer levels to the highest mean value and presented as % with SEM. Students t-test was performed to determine data significance.
All procedures of animal work were performed according to international recognized guidelines and the animal experimental protocols approved by ‘The Experimental Animal Inspectorate in Denmark’ under The Danish Veterinary and Food Administration, Ministry of Food, Agriculture and Fisheries (Registration number: 2013 - 15 - 2934 - 00789-C2, issue date: March 5, 2013).
Results and discussion
Blood circulation profiles (Figure 2) constructed from blood plasma fluorescent polymer levels showed a Mw-dependent prolonged blood retention of the copolymers. At 24 h, the 54 kDa copolymer was still retained in the blood at ~10% and accordingly displayed a significant (P < 0.03) higher bioavailability (area under the curve, AUC = 698% · h) compared to 36 kDa (AUC = 313% · h) and 15 kDa (AUC = 45% · h) polymers [see Additional file 1: Figure S1]. First order clearance kinetics were modeled from 1 h post i.v. injection of dye-labeled copolymers of different Mw and exhibited a half-life of < 20 min for polymers of 15 kDa that increased up to 2.8 h for 36 kDa and 6.4 h for 54 kDa polymers, in general accordance with previous studies on HPMA copolymers . Renal filtration commonly occurs for polymers with a hydrodynamic radius below 5 nm that corresponds to ~30 to 50 kDa Mw, however, with renal clearance of linear polymers, such as in this work, being up to 10 times higher than those of more globular-shape . In this work, the highest Mw p(HPMA-co-AzMA) did not seem to be excessively larger than the renal clearance limit, but still exhibiting prolonged circulation, and was, thus, selected for evaluation in arthritic mice.
Enhanced passive accumulation of the 54 kDa dye-grafted p(HPMA-co-AzMA) in the joints of arthritic mice compared to healthy was demonstrated (Figure 3 and Figure 4) with the bioavailability in arthritic tissue (AUC = 1783% · h) being ~12 times higher (P = 0.01) compared to healthy tissue (AUC = 148% · h) (see Additional file 2: Figure S2). Additionally, at 24 h, polymer levels in the blood of arthritic animals were lower than that of healthy (see Additional file 3: Figure S3), but showed a higher level in arthritic paws compared to healthy (Figure 3 and Figure 4). This suggests the polymer to be escaping blood circulation faster in the arthritic mice, probably due to accumulation in the arthritic joints. The polymer levels in healthy mice joints initially increased by 63% within 10 minutes but subsequently quickly declined, probably as a result of blood clearance of the polymer in general and a healthy microvasculature not exhibiting permeability and polymer extravasation effects. Free dye in arthritic or non-arthritic tissue was rapidly excreted and almost cleared before the first time point at 10 min (Figure 3, 10 min).
The arthritic scores, as an indicator of inflamed severity, were not entirely uniform with individual mice exhibiting combinations of joint scores (1–4). A Spearman test of polymer accumulation (at 24 h) and arthritic score correlation was, therefore, performed (Figure 5), supporting strong correlation between polymer accumulation and the arthritic condition (r = 0.89, P < 0.0001).
In summary, this work demonstrates the in vivo characteristics of a new azide containing copolymer, (p(HPMA-co-AzMA)), which offers a more versatile and well-defined alternative to existing bioconjugate systems. Attractive in vivo properties such as prolonged blood retention and specific accumulation in inflamed joints of RA illustrate the potential of the material for systemic delivery of anti-inflammatory drugs for local effects in the treatment of RA.
O’Dell JR: Therapeutic Strategies for Rheumatoid Arthritis. N Engl J Med. 2004, 350: 2591-2602. 10.1056/NEJMra040226.
Arend WP: Physiology of cytokine pathways in rheumatoid arthritis. Arthritis Rheum-Arthritis Care Res. 2001, 45: 101-106. 10.1002/1529-0131(200102)45:1<101::AID-ANR90>3.0.CO;2-7.
Howard KA, Paludan SR, Behlke MA, Besenbacher F, Deleuran B, Kjems J: Chitosan/siRNA nanoparticle-mediated TNF-alpha knockdown in peritoneal macrophages for anti-inflammatory treatment in a murine arthritis model. Mol Ther. 2009, 17: 162-168. 10.1038/mt.2008.220.
Olsen NJ, Stein CM: New drugs for rheumatoid arthritis. N Engl J Med. 2004, 350: 2167-2179. 10.1056/NEJMra032906.
Wang D, Miller SC, Sima M, Parker D, Buswell H, Goodrich KC, Kope P: The arthrotropism of macromolecules in adjuvant-induced arthritis rat model: a preliminary study. Pharm Res. 2004, 21: 1741-1749.
Paleolog EM: Angiogenesis in rheumatoid arthritis. Arthritis Res. 2002, 4: S81-S90. 10.1186/ar575.
Koch AE: Angiogenesis as a target in rheumatoid arthritis. Ann Rheum Dis. 2003, 62: ii60-ii67.
Fang J, Nakamura H, Maeda H: The EPR effect: unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Adv Drug Deliv Rev. 2011, 63: 136-151. 10.1016/j.addr.2010.04.009.
Yuan F, Quan L-d, Cui L, Goldring SR, Wang D: Development of macromolecular prodrug for rheumatoid arthritis. Adv Drug Deliv Rev. 2012, 64: 1205-1219. 10.1016/j.addr.2012.03.006.
Choy EHS, Hazleman B, Smith M, Moss K, Lisi L, Scott DGI, Patel J, Sopwith M, Isenberg DA: Efficacy of a novel PEGylated humanized anti‒TNF fragment (CDP870) in patients with rheumatoid arthritis: a phase II double‒blinded, randomized, dose‒escalating trial. Rheumatology. 2002, 41: 1133-1137. 10.1093/rheumatology/41.10.1133.
Duncan R, Vicent MJ: Polymer therapeutics-prospects for 21st century: the end of the beginning. Adv Drug Deliv Rev. 2013, 65: 60-70. 10.1016/j.addr.2012.08.012.
Barz M, Luxenhofer R, Zentel R, Vicent MJ: Overcoming the PEG-addiction: well-defined alternatives to PEG, from structure–property relationships to better defined therapeutics. Polym Chem. 2011, 2: 1900-1918. 10.1039/c0py00406e.
Ebbesen MF, Schaffert DH, Crowley ML, Oupický D, Howard KA: Synthesis of click-reactive HPMA copolymers using RAFT polymerization for drug delivery applications. J Polym Sci A Polym Chem. 2013, 51: 5091-5099. 10.1002/pola.26941.
Wang D, Miller S, Liu X-M, Anderson B, Wang XS, Goldring S: Novel dexamethasone-HPMA copolymer conjugate and its potential application in treatment of rheumatoid arthritis. Arthritis Res Ther. 2007, 9: R2-10.1186/ar2106.
L-d Q, Yuan F, Liu X-m, Huang J-g, Alnouti Y, Wang D: Pharmacokinetic and biodistribution studies of N-(2-hydroxypropyl)methacrylamide copolymer-Dexamethasone conjugates in adjuvant-induced arthritis Rat model. Mol Pharm. 2010, 7: 1041-1049. 10.1021/mp100132h.
Khachigian LM: Collagen antibody-induced arthritis. Nat Protoc. 2006, 1: 2512-2516. 10.1038/nprot.2006.393.
Noguchi Y, Wu J, Duncan R, Strohalm J, Ulbrich K, Akaike T, Maeda H: Early phase tumor accumulation of macromolecules: a great difference in clearance rate between tumor and normal tissues. Jpn J Cancer Res. 1998, 89: 307-314. 10.1111/j.1349-7006.1998.tb00563.x.
Shiah JG, Dvořák M, Kopečková P, Sun Y, Peterson CM, Kopeček J: Biodistribution and antitumour efficacy of long-circulating N-(2-hydroxypropyl)methacrylamide copolymer–doxorubicin conjugates in nude mice. Eur J Cancer. 2001, 37: 131-139. 10.1016/S0959-8049(00)00374-9.
Leblond F, Davis SC, Valdés PA, Pogue BW: Pre-clinical whole-body fluorescence imaging: Review of instruments, methods and applications. J Photochem Photobiol B Biol. 2010, 98: 77-94. 10.1016/j.jphotobiol.2009.11.007.
Etrych T, Šubr V, Strohalm J, Šírová M, Říhová B, Ulbrich K: HPMA copolymer-doxorubicin conjugates: the effects of molecular weight and architecture on biodistribution and in vivo activity. J Control Release. 2012, 164: 346-354. 10.1016/j.jconrel.2012.06.029.
We thank the Lundbeck Foundation for supporting this work through the grant: Lundbeck Foundation Nanomedicine Center for Individualized Management of Tissue Damage and Regeneration.
The authors declare that they have no competing interests.
MFE and KAH provided conception of research and drafted the manuscript; MFE, KAH, KB and BWD, planned the study; MFE and KB performed experiments; MFE and BWD analyzed and interpreted data; MFE prepared figures. All authors read and approved the final manuscript.
Electronic supplementary material
Additional file 1: Figure S1: Blood plasma bioavailability (AUC), in healthy BALB/c mice, of p(HPMA-co-AzMA) of 15, 36 and 54 kDa calculated from blood plasma polymer profiles in Figure 2. (PDF 103 KB)
Additional file 2: Figure S2: Bioavailability (AUC) of dye-labeled p(HPMA-co-AzMA) of 54 kDa and free dye in arthritic and healthy joints. AUC’s are calculated from p(HPMA-co-AzMA) joint accumulation profiles in Figure 3. (PDF 106 KB)
Additional file 3: Figure S3: Blood plasma levels of dye-labeled p(HPMA-co-AzMA) and free dye at 24 h. Polymer levels in the blood indicate that the polymer escapes blood circulation faster in the arthritic mice possible due to accumulation in the arthritic mice joints. Levels are normalized to the highest mean value and averages shown with SEM. (PDF 92 KB)
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
To view a copy of this licence, visit https://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
About this article
Cite this article
Ebbesen, M.F., Bienk, K., Deleuran, B.W. et al. Extended blood circulation and joint accumulation of a p(HPMA-co-AzMA)-based nanoconjugate in a murine model of rheumatoid arthritis. Mol and Cell Ther 2, 29 (2014). https://doi.org/10.1186/2052-8426-2-29
- Collagen Antibody-Induced Arthritis
- Extended circulation
- in vivo
- Image analysis
- Joint accumulation